2 Material and Methods
2.2.3 Biochemical Techniques
polyclonal antibody that was added after washing with PBS (200 x g, 5 min). At least 10,000 KIR‐AcGFP positive cells were recorded using a flow cytometer (LSR II) and analysed using FlowJo 8.8.7 software.
2.2.2.6 Antibody staining of PBMC for flow cytometry
Different leukocyte markers were used for the staining of PBMC and analysis of KIR
expression of different cell populations. For each sample 1‐2 x 106 cells were stained as shown in Table 2 and different anti‐macaque KIR antibodies were used. The PBMC samples were incubated with the according antibody mixture for 30 min at 4 °C, fixed with 3.5 % formaldehyde in FACS‐buffer for 10 min at RT and centrifuged for 5 min (200 x g). The cell pellets were resuspended in 50 µl FACS‐buffer, measured on a LSR II and further analysed using FlowJo 8.8.7 software.
Table 2. Gating strategy for multi‐colour flow cytometry
KIR ‐ NK cell I KIR ‐ NK cell II KIR ‐ T cell
FITC ‐ CD56 TCR g/d
PE CD159a CD159a CD159a
PerCP‐Cy5.5 CD14 CD14 CD14
PE‐Cy7 CD20 CD20 CD20
APC ‐ ‐ ‐
Alexa700 CD3 CD3 CD3
APC‐Cy7 CD16 CD16 CD16
Alexa450 ‐ ‐ CD4
V500 CD8 CD8 CD8
2.2.3 Biochemical Techniques
2.2.3.1 SDS‐PAGE
For the separation of proteins in a SDS gel the samples were mixed with 1 volume of Laemmli buffer and 1 volume DTT and incubated for 5 min at 95 °C. Then, the samples were transferred to a 10 % SDS gel and run under reducing conditions for about 1 h at 30 mA.
Following the separation of protein samples in a SDS gel the proteins were transferred to a nitrocellulose membrane by tank blotting for 1 h. Afterwards, the membrane was blocked with 5 % milk powder dissolved in TBS for about 1 h, washed three times with TBS followed by the addition of antibody over night. After three washing steps the membrane was incubated with a second antibody for 1 h and washed again with TBS five times. The membrane was developed by mixing the two developing solutions 1:1 followed by the incubation of the membrane for 1 min. The membrane was exposed to a Hyperfilm for varying time periods depending on the intensity of the detected signal.
The film was developed using a film processor.
2.2.3.3 Protein purification
The supernatant of either KIR‐Fc fusion protein expressing HEK293 cells or antibody producing hybridoma cells that were both grown in serum‐free ultraCHO medium for three days was collected, centrifuged (10 min, 200 x g) and filtered (0.45 µm) before purifying it using a protein G sepharose column. The column was equilibrated with 5 ml distilled H2O followed by 3 ml of binding buffer. Afterwards, the supernatant was passed through the column, eluted with 8 ml elution buffer and the pH of the eluate was neutralised with 75 µl per ml neutralising buffer. For further applications the eluate was concentrated using Amicon Ultra‐30 centrifugal filter units.
2.2.3.4 Quantification of protein concentrations
The protein concentration was measured using a spectrophotometer according to the manufacturer’s recommendation.
2.2.3.5 Enzyme‐linked immunosorbent assay (ELISA)
ELISA plates were coated over night at 4 °C with 0.5 µg of antigen diluted in 100 µl of coating buffer. After washing three times with PBS the plate was blocked with 4 % milk powder in PBS for 1 h at room temperature. Subsequently, the plate was washed for three times with PBS before the incubation with the first antibody for 1 h at 4 °C. As
negative control only PBS was added. The plate was washed three times with PBS and incubated with the second HRP‐conjugated antibody. After at least three times of washing the substrate reaction was added and incubated for around 10 min before being read at OD 405 nm in the ELISA reader.
2.2.3.6 Immunisation of mice with antigen
C3H/HeN or C57BL/6 mice were immunised with 100 µg KIR‐Fc protein as antigen.
The first immunisation was performed using Titermax Gold (Sigma) by subcutaneous injection followed by two intra‐peritoneal injections and a final boost by intravenous injection only using the antigen. Blood samples were collected before the first and after the third injection.
2.2.3.7 Fusion of cells
X63Ag8.653 mouse myeloma cells were used and fused to isolated mouse spleen cells that were prepared by Prof. Dr. Ralf Dressel (University of Göttingen). Generation, selection and cloning of hybridoma cells were performed using the ClonaCell‐HY Hybridoma Kit following the manufacturer’s protocol (Figure 5). Antibody‐secreting hybridoma significantly binding the coated appropriate antigen and not the coated human IgG using indirect ELISA, were selected and cultured in the presence of DMEM/20% fetal calf serum/1% penicillin/streptavidin.
2.2.3.8 Antibody labelling
The purified antibodies were first dialysed against PBS over night at 4 °C using Slide‐A‐
Lyzer dialysis units from Thermo Scientific to remove the elution buffer and to create better conditions for the conjugation with a fluorochrome. Afterwards, the labelling of purified and dialysed antibodies was performed using the DyLight Fluor Antibody Labeling Kit according to the manufacturer’s recommendation.
Figure 5. ClonaCell‐HY procedure overview.
Step 1: Fusion of spleen cells with X63Ag8.653 mouse myeloma cells. Step 2: Culturing and selection of cells in methylcellulose‐based medium containing HAT for around 14 days. Step 3: Harvesting of colonies derived of single cells. Step 4: Screening of hybridoma clones for antigen specificity using ELISA. Step 5: Expansion of selected hybridoma clones for the production of monoclonal antibodies (figure taken from ClonaCell‐HY, hybridoma cloning kit technical manual, Stemcell Technologies).
3 Results
3.1 Characterisation of monoclonal anti‐rhesus macaque KIR antibodies
Monoclonal antibodies are important tools to detect or study the protein expression of certain molecules and to help to purify them. They are used in biochemistry and molecular biology but also in medicine where monoclonal antibodies are applied for therapy. The first and most important step when working with antibodies is their characterisation. The antigen specificity and the methodical application need to be determined. Depending on whether a specific epitope is linear or conformational, certain antibodies are only suitable for specific methods.
Anti‐rhesus macaque KIR antibodies were generated by using KIR‐Fc fusion proteins to immunise mice and also for the first screening steps of the newly generated hybridoma clones. Supernatants of around 1700 clones were checked for reactivity with the respective KIR protein used for immunisation and with human IgG using ELISA. IgG‐
reactive supernatants were excluded from further analysis. The anti‐KIR antibody producing hybridomas were further characterised for their ability to cross‐react with other KIR molecules than their specific antigen using ELISA, their applicability in immunoblot analyses, the recognition of KIR molecules expressed by transfected HEK293 cells and finally, the recognised epitope was determined.
3.1.1 Establishment of anti‐rhesus macaque KIR antibodies
For the generation of monoclonal anti‐rhesus macaque KIR antibodies mice were immunised with KIR‐Fc fusion proteins (Rosner et al., 2011). Before the final boost with antigen, serum was taken and compared with pre‐immunisation serum using ELISA. The ELISA plates were coated either with the appropriate antigen or human IgG to exclude mice only producing antibodies against human IgG, because of the Fc portion, which is part of the fusion protein. All mice used for the generation of antibodies turned out to be reactive against the antigen and after the final